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Analytica Chimica Acta 568 (2006) 200–210

Review Microbial Yu Lei a,∗, Wilfred Chen b, Ashok Mulchandani b,∗ a Division of Chemical and and Centre of , Nanyang Technological University, Singapore 637722, Singapore b Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA Received 29 August 2005; received in revised form 17 November 2005; accepted 21 November 2005 Available online 18 January 2006

Abstract A microbial is an analytical device that couples microorganisms with a to enable rapid, accurate and sensitive detection of target analytes in fields as diverse as medicine, environmental , defense, food processing and safety. The earlier microbial biosensors used the respiratory and metabolic functions of the microorganisms to detect a substance that is either a substrate or an inhibitor of these processes. Recently, genetically engineered microorganisms based on fusing of the lux, gfp or lacZ reporters to an inducible gene promoter have been widely applied to assay and bioavailability. This paper reviews the recent trends in the development and application of microbial biosensors. Current advances and prospective future direction in developing microbial biosensor have also been discussed. © 2005 Published by Elsevier B.V.

Keywords: Microbial biosensors; Amperometric; Potentiometric; Optical; Luminescence;

Contents

1. Introduction ...... 201 2. Advantages of using microorganisms as biosensing elements ...... 201 3. Immobilization of microorganisms ...... 201 3.1. Chemical methods ...... 201 3.2. Physical methods ...... 201 4. Electrochemical microbial biosensor ...... 202 4.1. Amperometric microbial biosensor ...... 202 4.2. Potentiometric microbial biosensor ...... 204 4.3. Conductimetric biosensor ...... 205 4.4. Microbial fuel type biosensor...... 205 5. Optical microbial biosensor ...... 205 5.1. Bioluminescence biosensor ...... 205 5.2. Fluorescence biosensor ...... 207 5.2.1. Green fluorescence -based biosensor...... 207 5.2.2. O2-sensitive fluorescent material-based biosensor ...... 207 5.3. Colorimetric biosensor ...... 207 6. Other types of microbial biosensors ...... 208 6.1. based on baroxymeter for the detection of pressure change ...... 208 6.2. Sensors based on infrared analyzer for the detection of the microbial respiration product CO2 ...... 208 7. Future trends ...... 208 Acknowledgements ...... 208 References ...... 208

∗ Corresponding authors. Tel.: +65 67906712; fax: +65 67947553. E-mail addresses: [email protected] (Y. Lei), [email protected] (A. Mulchandani).

0003-2670/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.aca.2005.11.065 Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 201

1. Introduction ers with a close proximity. Since microbial biosensor response, operational stability and long-term use are, to some extent, a A biosensor is an analytical device that combines a bio- function of the immobilization strategy used, immobilization logical sensing element with a transducer to produce a technology plays a very important role and the choice of immo- proportional to the analyte concentration [1–18]. This signal bilization technique is critical. Microorganisms can be immobi- can result from a change in protons concentration, release lized on transducer or support matrices by chemical or physical or uptake of , light emission, absorption and so forth, methods [1–6]. brought about by the metabolism of the target compound by the biological recognition element. The transducer converts this 3.1. Chemical methods biological signal into a measurable response such as current, potential or absorption of light through electrochemical or Chemical methods of microbe immobilization include cova- optical means, which can be further amplified, processed and lent binding and cross-linking [1–6,24]. Covalent binding meth- stored for later analysis [1–3]. ods rely on the formation of a stable covalent bond between Biomolecules such as , , receptors, functional groups of the microorganisms’ cell wall components and microorganisms as well as animal and plant such as amine, carboxylic or sulphydryl and the transducer such cells or tissues have been used as biological sensing elements. as amine, carboxylic, epoxy or tosyl. To achieve this goal, whole Among these, microorganisms offer advantages of ability to cells are exposed to harmful chemicals and harsh reaction con- detect a wide range of chemical substances, amenability to dition, which may damage the cell membrane and decrease the genetic modification, and broad operating pH and tempera- biological activity. How to overcome this drawback is still a ture range, making them ideal as biological sensing materials challenge for immobilization through covalent binding. To our [1–18]. Microorganisms have been integrated with a variety of knowledge, this method has therefore not been successful for such as amperometric, potentiometric, calorimetric, immobilization of viable microbial cells [1–17,24]. conductimetric, colorimetric, luminescence and fluorescence to Cross-linking involves bridging between functional groups construct biosensor devices [1–8]. Several reviews papers and on the outer membrane of the cells by multifunctional reagents book chapters addressing microbial biosensor development have such as glutaraldehyde and cyanuric chloride, to form a net- been published [1–20]. The intent of this review is to highlight work. Because of the speed and simplicity, the method has found the advances in the rapidly developing area of microbial biosen- wide acceptance for immobilization of microorganisms. The sors with particular emphasis to the developments since 2000. cells may be cross-linked directly onto the transducer surface or on a removable support membrane, which can then be placed 2. Advantages of using microorganisms as biosensing on the transducer [1–17,24]. The ability to replace the membrane elements with the immobilized cells is an advantage of the latter approach. While cross-linking has advantages over covalent binding, the Enzymes are the most widely used biological sensing ele- cell viability and/or the cell membrane biomolecules can be ment in the fabrication of biosensors [1–4]. Although puri- affected by the cross-linking agents. Thus cross-linking is suit- fied enzymes have very high specificity for their substrates able in constructing microbial biosensors where cell viability is or inhibitors, their application in biosensors construction may not important and only the intracellular enzymes are involved in be limited by the tedious, time-consuming and costly the detection [8]. purification, requirement of multiple enzymes to generate the measurable product or need of cofactor/coenzyme. Microor- 3.2. Physical methods ganisms provide an ideal alternative to these bottle-necks [15]. The many enzymes and co-factors that co-exist in the and entrapment are the two widely used physical cells give the cells the ability to consume and hence detect methods for microbial immobilization. Because these meth- large number of chemicals; however, this can compromise the ods do not involve covalent bond formation with microbes and selectivity. They can be easily manipulated and adapted to provide relatively small perturbation of microorganism native consume and degrade new substrate under certain cultivating structure and function, these methods are preferred when viable condition [21–23]. Additionally, the progress in molecular biol- cells are required [8,14–17,24]. ogy/recombinant DNA technologies has opened endless possi- Physical adsorption is the simplest method for microbe bilities of tailoring the microorganisms to improve the activity immobilization. Typically, a microbial suspension is incubated of an existing enzyme or express foreign enzyme/protein in host with the or an immobilization matrix, such as alu- cell [19,20]. All of these make microbes excellent biosensing mina and glass bead [4,8,24], followed by rinsing with buffer elements. to remove unadsorbed cells. The microbes are immobilized due to adsorptive interactions such as ionic, polar or 3. Immobilization of microorganisms bonding and hydrophobic interaction. However, immobilization using adsorption alone generally leads to poor long-term stabil- The basis of a microbial biosensor is the close contact ity because of desorption of microbes. between microorganisms and the transducer. Thus, fabrication The immobilization of microorganisms by entrapment can of a microbial biosensor requires immobilization on transduc- be achieved by the either retention of the cells in close 202 Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 proximity of the transducer surface using or filter can determine BOD of a specific waste, research efforts have membrane or in chemical/biological polymers/ such as also been directed at improving the amperometric transducer (alginate, carrageenan, agarose, chitosan, collagen, polyacry- itself. For example, a miniaturized oxygen electrode based on lamide, polyvinylachohol, poly(ethylene glycol), polyurethane, thick-film screen-printing was recently developed to replace etc. [1–4,8,15]. A major disadvantage of entrapment immo- the bulky Clark dissolved oxygen electrode transducer. The bilization is the additional diffusion resistance offered by the widely used thick-film screen-printing technique was used to entrapment material, which will result in lower sensitivity and print the platinum-working electrode, Ag/AgCl reference elec- detection limit. trode and platinum auxiliary electrode of the amperometric Microbial biosensor can be classified based on the transduc- oxygen electrode on an inert substrate. The oxygen electrode ers into electrochemical, optical and others. was then modified with A. adeninivorans LS3 by entrapment in poly(carbamoyl) sulfonate (PCS) and successfully applied 4. Electrochemical microbial biosensor for rapid (∼100 s) and stable (up to 2 months) BOD deter- mination [26]. Similarly, to extend the dynamic range of the There are three types of electrochemical microbial biosen- BOD , which in the case of dissolved oxygen electrode is sors: amperometric, potentiometric, and conductometric [1–6]. limited by the solubility of oxygen in the sample, a ferricyanide- mediated microbial biosensor using a novel yeast strain for 4.1. Amperometric microbial biosensor BOD measurement was developed [58]. Recently, an amper- ometric transducer array featuring four individually addressable Amperometric microbial biosensor operates at fixed potential platinum was constructed and modified with two with respect to a reference electrode and involves the detection microbial strains with different substrate spectra for the mea- of the current generated by the oxidation or reduction of species surement of BOD and poly cyclic aromatic hydrocarbons (PAH) at the surface of the electrode. Table 1 summarizes a few of the simultaneously [28]. amperometric biosensors reported in the literature. Besides BOD biosensor, amperometric microbial biosen- Amperometric microbial biosensors have been widely devel- sors have also been applied for measurement of several other oped for the determination of biochemical oxygen demand chemicals. Because of its importance in indus- (BOD) for the measurement of biodegradable organic pollutants try and clinical toxicology [8,59], microbial biosensors for in aqueous samples [25]. The conventional standard method for ethanol has garnered the second most research attention after the determination of BOD measures the microorganisms’ oxy- BOD. Different microorganisms metabolizing ethanol such as gen consumption/respiration over a period of 5 days [26,27] and Trichosporon brassicae [60], Acetobacter aceti [61], Candida is reported as BOD5 [25,28]. While BOD5 is a good indicator of vini [62], Gluconobacter suboxydans [63], C. tropicalis [64], the concentration of organic pollutants in water, it is extremely [65], Saccharomyces ellipsoideus [66], G. slow and hence not suitable for process control [29,30].To oxydans [59] and Pichia methanolica [59] have been immo- address this limitation, several BOD biosensors based on amper- bilized on oxygen electrode to fabricate ethanol biosensors. ometric oxygen electrode transducer modified with microor- While these biosensors posses good sensitivity and stability, ganisms degrading/metabolizing organic pollutants have been they usually have poor selectivity. Thus, there is a great interest reported [25–30]. The microbial strains used as biological sens- to develop selective microbial ethanol biosensor. An improved ing element include Torulopsis candida [31], Trichosporon cuta- selectivity for ethanol determination in presence of was neum [32,33], Pseudomonas putida [34], Klebsiella oxytoca achieved by replacing oxygen with ferricyanide as the elec- AS1 [35], Bacillus subtilis [36,37], Arxula adeninivorans LS3 tron acceptor mediator for G. oxydans immobilized on a glassy [38–41], Serratia marcescens LSY4 [42], Pseudomonas sp. carbon electrode by cellulose acetate membrane which also [43], P. fluorescens [44,45], P. putida SG10 [46], Thermophilic restricted the availability of glucose to the cells by size exclusion [47], Hansennula anomala [48] and yeast [29]. Because [67,68]. any given strain provides a narrow substrate spectrum, single- Sugars are important ingredients of different media and sen- strain-BOD-biosensor has limitations in analyzing complex sors for determination of sugars are therefore highly desired. samples. This bottleneck can be alleviated by employing a mix- Microbial biosensors for sugars have ranged from the simple ture of two or more microorganisms to broaden the substrate and modification of Clark and microfabricated oxygen electrode hence analyte spectrum with a stable performance [49–57]. with S. cerevisiae and E. coli K12 mutants, respectively, to mod- As the most extensively investigated microbial biosensor, ification of graphite electrode with G. oxydans in conjunction the first commercial BOD biosensor was produced by Nisshin with hexacyanoferrate (III) as a mediator [69–71]. Denki (Electric) in 1983. Since then, several more BOD biosen- Phenol and substituted phenols have received considerable sors have been commercialized by DKK Corporation, Japan; attention in waste analysis program due to their high toxic- Autoteam FmbH, Germany; Prufgeratewerk Medingen GmbH, ity to mammals, humans and plants. A variety of amperomet- Germany; Dr. Lange GmbH, Germany; STIP Isco GmbH, Ger- ric microbial biosensors [72–78] have been reported for these many; Kelma, Belgium; LAR Analytik and Umweltmesstechnik EPA Priority chemicals. p-Nitrophenol (PNP) degrading bacte- GmbH, Germany; Bioscience, Inc., USA; USFilter, USA [8,25]. rial Arthrobacter JS 443 and Moraxella sp. isolated from PNP While most of the research and development in BOD biosen- contaminated sites in the U.S. have been immobilized on oxy- sors has focused in identifying different microorganisms that gen and carbon paste electrodes using polycarbonate membrane Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 203

Table 1 Amperometric microbial biosensors Target Microorganism Limit of detection References BOD A. adeninivorans LS3 1.24 mg/l [26] BOD C. parapsilosis 1 mg/l [28] BOD yeast 1 mg/l [29] BOD T. cutaneum and B. subtilis 0.5 mg/l [30] BOD T. candida 7–75 ppm [31] BOD T. cutaneum 0–32 mg/l [32] BOD T. cutaneum 10–70 mg/l [33] BOD P. putida 0.5 mg/l [34] BOD K. oxytoca AS1 <44 mg/l [35] BOD B. subtilis (heat killed) 10–80 mg/l [36] BOD B. subtilis 2–22 mg/l [37] BOD A. adeninivorans LS3 8–550 mg/l [38] BOD A. adeninivorans LS3 2 mg/l [39] BOD A. adeninivorans LS3 – [40] BOD A. adeninivorans LS3 2.61 mg/l [41] BOD Serratia marcescens LSY4 0–44 ppm [42] BOD Pseudomonas sp. 1–40 mg/l [43] BOD P. fluorescens 15–200 mg O/l [44] BOD P. fluorescens 15–260 mg/l [45] BOD P. putida SG10 1 mg/l [46] BOD T. bacteria <10 mg/l [47] BOD BODSEED 5–45 mg/l [49] BOD Rhodococcus erythropolis DSM Nr. 772 and Issatchenkia – [50] orientalis DSM Nr. 3433 BOD B. subtilis and B. licheniformis 7B 10–70 mg/l [51] BOD B. subtilis and B. licheniformis 7B 0–80 mg/l [52] BOD B. subtilis and B. licheniformis 7B 0–70 mg/l [53] BOD Microbial consortium 5.0 mg BOD5/l [55,56] BOD M. consortium 1 mg/l [57] BOD Yeast SPT1 and SPT2 2 mg/l [58] Ethanol G. oxydans or P. methanolica 0.05 mM [59] Ethanol A. aceti (IFO 3284) <0.2 mM [61] Ethanol C. vini 0.02–0.2 mM [62] Ethanol G. suboxydans 0–25 mg/l [63] Ethanol C. tropicalis 0.5–7.5 mM [64] Ethanol A. niger 1–32 ppm [65] Ethanol S. ellipsoideus 69 ␮M [66] Ethanol G. oxydans 0.85 ␮M [67,68] Total sugars G. oxydans 1.1–2.2 g/l [69] Sucrose S. cerevisiae 6–100 mM [70] Mono-and /disaccharides E. coli K12 0–4 mM for disaccharides 0–2.5 mM for monosaccharide [71] p-Nitrophenol A. JS 443 5 nM [72] p-Nitrophenol A. JS 443 0.2 ␮M [73] p-Nitrophenol M. sp. 0.1 ␮M [74] p-Nitrophenol M. sp. 20 nM [75] 2,4-Dinitrophenol R. erthropolis 2–20 ␮M [76] Phenolic compounds P. putida 0.5–6 ␮M, 0.3–2.5 ␮M and 0.02–0.2 ␮M [77] Phenolic compounds P. putida 0.1–1.0 ␮M and 0.05–1.0 ␮M [78] Organophosph ates Recombinant Moraxella 0.2 ␮M paraoxon and 1 ␮M methyl parathion [80] Organophosph ates Recombinant P. putida JS 444 55 ppb of paraoxon, 53 ppb of methyl parathion, and [81] 58 ppb of parathion Cyanide S. cerevisiae 0.15 ␮M [82] Cyanide T. ferrooxidans 0.5 ␮M [83] Cyanide P. fluorescents NCIMB 11764 0.05–1 mg/l [86] Cyanide S. cerevisiae IFO 0377 0–15 ␮M [87] Cyanide S. cerevisiae 0.3–150 ␮M [88] Anionic surfactants Pseudomonas and Archromobacter 1 ␮lM [89] Non-ionic surfactants Comamonas testosterone TI 0.25 mg/l [90] Hydrogen peroxide A. peroxydans 0.1–9.5 ␮M [91] Acetic acid F. solani 2–70 ppm (v/v) [92] Microbiologic-ally P. sp. 0–0.7 mM sulphuric acid [93] influenced corrosion Cu2+ Recombinant S. cerevisiae 0.5–2 mM [95] Cadmium Recombinant E. coli 25 nM [96] 204 Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210

[73,74] and Nafion [72], respectively, and by directly mixing immobilized cell loading [93]. The same group also used Ace- in the carbon paste [75] to fabricate biosensor for PNP. Other tobacter sp. to develop amperometric microbial biosensor for microbial biosensors for phenols include Rhodococcus erthro- monitoring microbiologically influenced corrosion caused by polis modified Clark oxygen electrode for 2,4-dinitrophenol fungal species [94]. (2,4-DNP) [76] and P. putida DSM 50026, a well-known phe- Another application of amperometric microbial biosensors nol degrading microorganisms, modified thick-film and screen- is the detection of heavy metal for environmental con- printed graphite electrodes for phenols [77,78]. trol. A microbial biosensor to detect Cu2+ by an amperometric Neurotoxic (OP) compounds have found method has been developed using recombinant S. cerevisiae wide applications as and insecticides in containing with Cu2+-inducible promoter fused to the and as chemical warfare agents in military practice [8,79–81]. lacZ gene. In the presence of Cu2+, the recombinant strains Amperometric biosensors based on genetically engineered are able to utilize lactose as a carbon source and lead to the Moraxella sp. and P.putida with surface-expressed organophos- oxygen consumption change, which can be detected by using phorus hydrolase (OPH) have been developed for sensitive, oxygen electrode [95]. A novel promoter-based electrochemical selective and cost-effective detection of OPs. These biosen- biosensor for on-line and in situ monitoring of gene expres- sors relied on the amperometric detection of PNP generated sion in response to cadmium has also been described [96]. from hydrolysis of OP compounds by surface-displayed OPH A cadmium-responsive promoter from E. coli was fused to a or oxygen consumed and electrochemically active intermedi- promoterless lacZ gene, and then the ␤-galactosidase activ- ates formed during the further mineralization of the PNP by the ity was monitored using screen-printed electrode in the pres- cells [80,81]. ence of cadmium [96]. This whole-cell biosensor could detect The inhibition of bacterial respiration and hence the decrease nanomolar concentrations of cadmium on-line or in-site within of oxygen consumption rate, has been utilized to fabricate minutes. cyanide biosensor [82,83]. Whole-cell biosensors consisting of dissolved oxygen electrode modified with Nitrosomonas 4.2. Potentiometric microbial biosensor europaea, Thiobacillus ferrooxidans, Saccharomyces cerevisiae and Pseudomonas fluorescens were reported for batch and con- Conventional potentiometric microbial biosensors consist of tinuous cyanide monitoring [83–88]. an -selective electrode (pH, ammonium, chloride and so on) Other amperometric microbial biosensors based on mon- p p or a -sensing electrode ( CO2 and NH3 ) coated with an itoring of cell respiration include biosensor for surfactants, immobilized microbe layer. Microbe consuming analyte gen- representing a widespread group of organic pollutants, using erates a change in potential resulting from ion accumulation surfactant-degrading bacteria [89,90], hydrogen peroxide by or depletion. Potentiometric transducers measure the difference coupling immobilized living Acetobacter peroxydans [91] and between a working electrode and a reference electrode, and the for acetic acid using Fusarium solani [92]. signal is correlated to the concentration of analyte [2,3,16]. Due Over the last two decades, the microbiologically influenced to a logarithmic relationship between the potential generated corrosion (MIC) of metallic materials has received great atten- and analyte concentration, a wide detection range is possible. tion. A stable, reproducible and specific microbial biosensor was However, this method requires a very stable reference electrode, developed for monitoring MIC of metallic materials in industrial which may be a limitation of these transducers. A few examples systems based on Pseudomonas sp. isolated from corroded metal of biosensors based on potentiometric transducers are summa- surface and immobilized on acetylcellulose membrane at oxygen rized in Table 2. electrode. A linear relationship between the biosensor response The simplest potentiometric microbial biosensor is based and the concentration of sulfuric acid (the most corrosive inor- on the modification ion selective electrode. Several microbial ganic acid involved in microbial corrosion) was established. The biosensors based on modification of glass pH electrode with biosensor response time was 5 min and was dependent on many genetically engineered E. coli expressing organophosphorus parameters such as pH, temperature, corrosive environment and hydrolase intracellularly and on the outer surface of cells and

Table 2 Potentiometric microbial biosensors Target Microorganism Transducer type Limit of detection References

Organophosphates Flavobacteium sp. pH electrode 0.025–0.4 mM [97] Recombinant E coli pH electrode 2 ␮M [98] Organophosphates Recombinant E coli pH electrode 3 ␮M [99] Penicillin Recombinant E coli Flat pH electrode 5–30 mM [100] Penicillin Recombinant E coli pH electrode 1–16 mM [101] Tryptophan E coli WP2 LAPS 0–12 ␮M [102] + Urea Bacillus sp. NH4 ion selective electrode 0.55–550 ␮M [103] Trichloroethylene P. aeruginosa JI104 Chloride ion selective electrode 0.03–2 mg/l [104] Trichloroethylene P. aeruginosa JI104 Chloride ion selective electrode 0.1–4 mg/l [105] Ethanol S. ellipsoideus Oxygen 0.02–50 mM [106] Sucrose S. cerevisiae Oxygen 3.2 ␮M [107] Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 205 wild-type OP degrading bacteria Flavobacteium sp. have been cell as BOD sensor was improved using respiratory inhibitors reported [16,97–99]. The principle of detection is based on the [115]. detection of the protons released by OPH catalyzed hydrolysis of OP and correlating to the concentration of OPs. Similarly, 5. Optical microbial biosensor recombinant E. coli harboring the plasmids encoding for ␤- lactamase [100] and penicillinase [101] synthesis immobilized The modulation in optical properties such as UV–vis absorp- on pH electrode using gluten and acetylcellulose membranes tion, bio- and chemi-luminescence, reflectance and fluorescence entrapment, respectively, were developed for monitoring peni- brought by the interaction of the biocatalyst with the target ana- cillin [100,101]. A new type of solid state silicon-based light lyte is the basis for optical microbial biosensors [1–5]. Optical- addressable potentiometric sensor for monitoring hydrogen ion based biosensors offer advantages of compactness, flexibility, was integrated to the auxotrophic bacteria E. coli WP2 (requiring resistance to electrical noise, and a small probe size. Some rep- tryptophan for its growth) to fabricate a potentiometric microbial resentative bioluminescence and fluorescence based microbial assay for tryptophan [102]. biosensors are listed in Table 3. While pH electrodes are the most widely applied ion selective electrode for microbial biosensors, other ion selective electrodes 5.1. Bioluminescence biosensor have also been utilized. For example, an ammonium ion selective electrode was coupled with urease-yielding Bacillus sp. isolated Bioluminescence is associated with the emission of light by from soil to develop a disposable microbial biosensor for mon- living microorganisms and it plays a very important role in real- itoring the presence of urea in milk [103]. Similarly, a chloride time process monitoring. The bacterial luminescence lux gene ion selective electrode was modified with TCE degrading bac- has been widely applied as a reporter either in an inducible terium Pseudomonas aeruginosa JI104 for TCE monitoring in or constitutive manner. In the inducible manner, the reporter batch and continuous modes in wastewaters [104,105]. lux gene is fused to a promoter regulated by the concentra- A potentiometric oxygen electrode with immobilized S. ellip- tion of a compound of interest. As a result, the concentration of soideus was also successfully used to produce a microbial the compound can be quantitatively analyzed by detecting the biosensor for the determination of ethanol with an extended bioluminescence intensity [19,20]. In the constitutive manner, response range [106]. Based on the same format, sucrose biosen- the reporter gene is fused to promoters that are continuously sor based on an immobilized S. cerevisiae was also described expressed as long as the organism is alive and metabolically [107]. active [19]. This kind of reporter is good for evaluating the total toxicity of contaminant. Both types of reporters have been shown 4.3. Conductimetric biosensor to be useful for biosensor development. Heavy metal-mediated toxicity in the environment is depen- Many microbe-catalyzed reactions involve a change in ionic dant on bioavailable metal concentrations. Bioluminescent species. Associated with this change is a net change in the con- microbial biosensors have been extensively investigated to mon- ductivity of the reaction solution. Even though the detection itor bioavailable metal. Ralstonia eutropha AE2515 was con- of solution conductance is non-specific, conductance measure- structed by transcriptionally fusing cnrYXH regulatory ments are extremely sensitive [1–3]. to the bioluminescent luxCDABE report system to fabricate a Recently, a single-use conductivity and microbial sensor were whole cell biosensor for the detection of bioavailable concentra- developed to investigate the effect of both species and concen- tion of Ni2+ and Co2+ in soil [116]. Several optical biosensors tration/osmolarity of anions on the metabolic activity of E. coli. consisting of bacteria that contain gene fusion between the reg- This hybrid sensing system combines physico-chemical and ulatory region of the mer operon (merR) and luxCDABE have biological sensing and greatly increases the ease with which been developed to quantitatively response to Hg2+. The mer comparative data could be assimilated [108]. promoter is activated when Hg2+ binds to MerR, then result the transcription of the lux reporter gene and subsequent light 4.4. type biosensor emission [117–122]. Bioavailable copper in soil is also moni- tored by using engineered P.fluorescens through mutagenesis of Microbial fuel cells (MFCs) have been studied as a BOD P. fluorescens containing copper-induced gene and Tn5::luxAB sensor for a long time. Since Karube et al. reported a BOD promoter probe transposon [123]. sensor based on MFC using the hydrogen produced by Clostrid- In order to monitor nutrients in an aquatic ecosys- ium butyricum immobilized on the electrode in 1977 [109],a tem, a biosensor for monitoring phosphorus bioavailablity variety of MFC BOD sensors with use of electron-mediator to Cyanobacteria (Synechococcus PCC 7942) was developed have been developed [110–112]. Even though the addition [124]. The reporter strain Synechococcus harbors the gene cod- of mediators in these biosensors can enhance the electron ing the reporter protein luciferase under the control of an transfer, these biosensors have poor stability because of the inducible alkaline phosphatase promoter, which can be induced toxicity of mediators. Recently, mediator-less microbial fuel under phosphorous limitation and shows improvement to con- cells have been exploited to fabricate novel BOD sensors for ventional phosphorus detection methods [124]. Bioluminescent continuous and real-time monitoring [113,114]. Furthermore, microbial biosensors using the inducible reporter gene have Kim et al. reported that the performance of a microbial fuel also been developed for the measurement of bioavailable naph- 206 Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 [143] [144] [141] [142] [138] [139] [135] [128] [129] [116] [125] [126] [127] [117] [131] [132] [133] [136] [140] [134] [130] [118] [119] [121] [122] [123] [124] [137] M ␮ 2+ values ranged from 0.09 to MCo ␮ 50 M ,9 13 2+ UV dose − 2 M 10 M toluene M in synthetic glucose medium and 1.5 7 values (mg/l) dependent on tested pollutants values (g/l) ranged from 0.034 to 0.638 MNi M ␮ × ␮ − ␮ ␮ 50 50 10 2 ppm ∼ 21 mg/l for different pillutants ∼ in LB medium Fluorescence 4 mg/l Fluorescence – Luminescence EC Fluorescence – Luminescence – Luminescence 50–500 nM Luminescence >100 mg/l Luminescence nM level melA genes gene Fluorescence gene cassette Luminescence On-line test EC ) Fluorescence – JL1157 (pTolLHB) Fluorescence 0.02 gfp luxAB strain Luminescence 0.02 gfp fusion Luminescence 1.2 J/cm egfp gene fused to the fusion Luminescence 100 ppb mitomycin cloacae carrying luxCDABE gfp pRB27 or pRB28 Luminescence 0.2 ng/g plasmid pRB27 or pRB28plasmid pRB27plasmid pRB28, Luminescence – Luminescence 10 pM plasmid pTOO11 Luminescence 1.67 Pseudomonas syringae luxAB promoter probe transposon Luminescence 0.3 ppm recA’::lux :: genes and E. colir::luxAB recA’::lux promoter and the mer-lux mer-lux mer-lux mer-lux mer-lux fusions Luminescence EC50 values (mg/l) dependent on tested pollutants sal containing a ::lux containing DL-2-haloacid dehalogenase containing ’ AE2515 Luminescence 0.1 luxCDABE PCC 7942 reporter strain Luminescence 0.3 fab A FRD1 carrying plasmid pMOE15 with A506 (pTolLHB)and E. pUCD607 Luminescence 10586r pUCD607 Luminescence EC DF57 with a Tn5 (pPR-arsR-ABS, expressing P. putida BS566::luxCDABE Pseudomonas syringae E. coli E. coli ␣ and and optical fiber sensor from ASR Co. Ltd. Fluorescence 0.5 mg/l carrying NAH7 plasmid and a chromosomally inserted JM-109 pQE60-EGFP Fluorescence – bearing MC1061 harboring DPD1718 containing DH5 MC4100 harboring pAHL-GFP Fluorescence – HMS174 harboring HMS174 harboring HMS174 harboring HMS174 harboring co//HB101 pUCD607 containing P. putida E. coll microorganisms P. fluorescens harbouring the fusion of proU promoter and E. coli P. fluorescens E. coli E. P.fischeri E. coli E. coli promoter E. coli, Pantoea agglomerans E. coli P. aeruginosa recA::luxCDABE P. fluorescens Ralstonia eutropha E. coli E. coli E. coli E. coli P. fluorescens Synechococcus P. putida gene fusion between the encoding gene and Sinorhizobium meliloh pOS14orpOS15 2+ and Co 2+ phenolics-containing waster 2+ -Acyl homoserine lactones in soil BOD Cell population BOD Oxygen-sensitive fluorescent material and sea water Bioavailable toluene and related compounds N Bioavailable iron Recombinant Pollutants/toxicity Pollution- induced stress Urinary mercury (II) TributyltinHalogenated organic acidsWater pollutants/toxicity Recombinant Bioluminescent recombinant Hg Toxicity of waster water treatment plant treating Genotoxicants UV Recombinant Arsenite Water availability UV Toxicity of chlorophenol Table 3 Bioluminescence and fluorescence microbial biosensors Bioavailable mercury Bioavailable mercury Bioavailable mercury Bioavailable copper Bioavailable phosphorus Bioavailable naphthalene TargetNi Microorganism Transducer type Limit of detection References Galactosides Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 207 thalene [125], tributyltin [126] and halogenated organic acids and not known to be produced by microorganism indigenous to [127]. terrestrial habits, it provides great advantage and flexibility when The environmental problems caused by industrial and agri- evaluating reporter activity. The primary disadvantage of GFP cultural pollution have increased the demand for the develop- as a reporter protein is the delay between protein production and ment of pollutant and toxicity detection methods. The fusion protein fluorescence. of reporter genes to promoters that are induced when cell are The GFP-based microbial biosensor has been shown to be stressed by toxic chemicals are one promising approach that has useful in assessing heterogeneity of iron bioavailability on plant been used to fabricate biosensor for such application. Recom- [136]. In this sensor, ferric iron availability to cells was assessed binant E. coli bearing fabA’::lux fusion and plasmid pUCD607 by quantifying the fluorescence intensity of cells containing a containing the full luxCDABE cassette have been constructed plasmid-borne transcriptional fusion between an ion-regulated as biosensors for water pollutant detection [128,129]. The on- promoter and gfp [136]. Recently, Wells et al. developed an line pollutant and toxicity test, using bioluminescence-based ultrasensitive biosensor for arsenite by using laser-induced fluo- biosensors, was proved to be sensitive and reliable. Lux-marked rescence confocal to measure arsenite-stimulated rhizobacterium P. fluorescens has been developed to evaluate enhanced green fluorescent protein synthesis of genetically the pollution-induced stress, which influences rhizobacterium engineered E. coli bioreporter cell, which has an inherent carbon flow based on the fact that bioluminescence output single-molecule detection capability [137]. A recombinant of biosensor is directly correlated with metabolic activity and soil bacterium Sinorhizobium meliloti has been constructed by reports on carbon flow in root exudates [130]. Furthermore, fusing the gfp gene to the melA promoter, which is induced on lux-marked whole cell biosensors for evaluation of interac- exposure to galactose and galactosides. Using this fusion strain, tive toxicity of chlorophenol [131] and toxicity assessment a biosensor was developed to determine the concentration of of a wastewater treatment plant treating phenolics-containing galactosides [138]. Similarly, gfp reporter gene has also been waste [132] have been reported, respectively. These biosensors used to develop biosensors for various applications, such as responded to tested pollutants fast and enable a rapid toxicity test detecting bioavailable toluene and related compounds [139] and possible. N-acyl homoserine lactones in soil [140], measuring water avail- Genotoxicants is a class of hazards, which can cause DNA ability in a microbial habitat [141], monitoring cell populations damage. An optical-fiber bioluminescent microbial sensor to [142] and so on. With the development of DNA recombinant detect the DNA damage hazard-mitomycin C by the induc- technologies and our understanding to microbes, this type tion of a selected promoter and the subsequent production of of biosensor will become an increasingly more powerful bioluminescent light through a recombinant lux reporter was technique. reported. Bioluminescence production was shown to be dose- dependent [133]. E. coli containing plasmid-borne fusion of the 5.2.2. O2-sensitive fluorescent material-based biosensor recA promoter-operator region to the Vibrio fischeri lux genes Besides green fluorescent protein, other fluorescent materials has also been reported for genotoxicant detection. When the have also been used in the construction of microbial biosen- recombinant E. coli strains are challenged with DNA damage sor. Recently, fiber-optical microbial sensors for determination hazards, they increase their luminescence [134]. Furthermore, of BOD were reported [143,144]. The biosensors consisted of this study was expanded by investigating and demonstrating either a layer of oxygen-sensitive fluorescent materials that are the luminescence response of these strains to radi- made up of seawater microorganisms immobilized in poly(vinyl ation, which can cause DNA damage [134]. Another lux-based alcohol) sol–gel matrix and an oxygen fluorescence quenching Psedumonoas aeruginosa biosensor was fabricated to quantify indicator with linear range of 4–200 mg/l [143], or an immo- bacteria exposure to UV radiation in biofilm [135]. bilized P. putida membrane attached to an optical fiber sensor for dissolved oxygen from ASR Co. Ltd. with detection limit of 5.2. Fluorescence biosensor 0.5 mg/l [144].

Fluorescence spectroscopy has been widely applied in ana- 5.3. Colorimetric biosensor lytical . It is a sensitive technique that can detect very low concentrations of analyte because of the instrumental prin- A sensitive biosensor based on color changes in the toxin- ciples involved. At low analyte concentrations, fluorescence sensitive colored living cells of fish was reported [145].In emission intensity is directly proportional to the concentration. the presence of toxins produced by microbial pathogens, the Fluorescent materials and green fluorescent protein have been cells undergo visible color change and the color changes in extensively used in the construction of fluorescent biosensor a dose-dependant manner. The results suggest this cell-based [1–5]. biosensor’s potential application in the detection and identifica- tion of virulence activity associated with certain air-, food-, and 5.2.1. Green fluorescence protein-based biosensor water-borne bacterial pathogens. Like bioluminescent reporter lux gene, gfp gene coding for the We reported a simple fiber-optic based microbial sensor green fluorescent protein (GFP) has also been widely applied as to detect organophosphates based on the absorbance of PNP reporters and fused to the host gene that allows reporter activity formed from the hydrolysis of organophosphates by the geneti- to be examined in individual cells. Because GFP is very stable cally engineered E. coli expressing organophosphorus hydrolase 208 Y. Lei et al. / Analytica Chimica Acta 568 (2006) 200–210 on the cell surface [146]. This biosensor can be easily extended microbial biosensor is still hampered because they suffer from to other organophosphates such as coumaphos through the mon- long response time, low sensitivity and poor selectivity. itoring of its hydrolysis product coumarin. With a better understanding of the genetic A colorimetric whole cell bioassay for the detection of com- of microbes and the development of improved recombinant mon environmental pollutants benzene, toluene, ethyl benzene DNA technologies, different enzymes and have been and xylene (BTEX), found at underground fuel storage tanks, expressed on the cell surface through surface expression using recombinant E. coli expressing toluene dioxygenase and anchors. In this format, the microbes can serve as an enzymes’ toluene dihydrodiol dehydrogenase was reported. The bioassay support matrix. The surface expressed enzymes or proteins can was based on the enzyme catalyzed conversion of the BTEX directly react with substrates without the entry of substrates into components to their respective catechols followed by the reac- the microbes. Through this way, faster response and highly sensi- tion with hydrogen peroxide in presence of horseradish perox- tive microbial biosensors can be developed [80,81,98,146]. The idase to colrimetric products that can be monitored at 420 nm same surface expression system can also be applied to produce [147]. other pollutant-resistant strains for biosensor fabrication. Because non-specific cellular response to substrates and 6. Other types of microbial biosensors intermediates of microbial catabolism can limit the selectivity of microbial biosensors, the ability to design, select and screen Besides electrochemical, optical and colorimetric microbial for microorganisms with specific activity for certain chemical biosensors, there are few other types of biosensors reported compounds will play an important role in the development of recently. high selectivity microbial biosensors. To achieve this goal, it is necessary to combine the classical knowledge in 6.1. Sensors based on baroxymeter for the detection of with the rapidly expanding methodologies in genetic engineer- pressure change ing in order to control or create microbe’s metabolic pathway (on/off) according to our purpose. As a new application, baroxymeter has been developed as a Another trend in microbial biosensors is to develop biosen- portable wastewater direct toxicity assessment device based on sors for the application in extreme conditions, such as highly manometric bacterial respirometry. Respirometry was measured acidic, alkaline, saline, extreme temperature and organic sol- as the pressure drop in the headspace of a close vessel due to vent environment because more and more detections will involve oxygen uptake by the microorganism in contact with sample. such unfriendly conditions. Since normal microbes can not sur- This microbial showed good reproducibility and vive in such harsh environment, the selection of microorganism, comparable responses with other reported methods [148]. which survive under these extreme conditions while maintaining high enzymes activities will become more and more important in 6.2. Sensors based on infrared analyzer for the detection of future development of microbial biosensor. These requirements the microbial respiration product CO2 are particularly important for the growing biosensor industry because of the great need for low cost, sensitive, selective and A new method was reported for monitoring inhibitory effects fast response biosensor in the market [151]. We believe, with in wastewater treatment plants based on continuous measure- current advances in microbial biosensor and progress in modern ment of the microbial respiration product CO2. Activated sludge biotechnology, microbial biosensors will have a promising and microbes are used as the biological elements and their respiratory bright future. activity is inhibited by the presence of toxic compounds, result- ing in a decrease in CO concentration which was analyzed by 2 Acknowledgements using a CO2 infrared analyzer [149]. Based on the measurement of CO concentration in the off gas produced during degradation 2 We greatly appreciate the support of U.S. EPA and USDA for of carbon compound by microbial respiration activities, a micro- supporting studies on microbial biosensors. bial biosensor was developed to monitor the extent of organic pollution in wastewater both off-line in a laboratory and online in a wastewater treatment plant [150]. References

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